Project Summary Virus-host interactions drive a remarkable diversity of host immune responses and viral countermeasures during host-pathogen coevolution. Identifying and characterizing virus-host interactions within this evolutionary ?arms race? is critical for understanding how the outcome of these interactions favor either productive pathogen replication or abortive infection. Furthermore, if one can identify pathogen-encoded immune evasion proteins (IEPs), they might be exploited to discover and probe the cellular machinery they target. We are developing new approaches to identify IEPs that target conserved eukaryotic machinery and are capable of tipping the balance between abortive and productive viral infection. While classical approaches have employed virus-host models wherein the virus can productively infect the chosen host, we have developed a new paradigm that exploits naturally abortive arbovirus infections in lepidopteran (moth and butterfly) cells as a screening tool to identify novel IEPs encoded by mammalian pathogens that convert abortive infections to productive infections by countering host immune responses. By identifying IEPs encoded by mammalian pathogens that retain immunosuppressive function in insect cells, we can select for IEPs that target antiviral machinery conserved between invertebrate and vertebrate hosts. Using this approach, we have identified several IEPs encoded by mammalian pathogens that target conserved host machinery that we have subsequently found to perform antiviral functions. For example, one current area of focus is the characterization of poxvirus-encoded A51R proteins as a new family of mammalian IEPs that target the facilitates chromatin transcription (FACT) complex, an evolutionarily-conserved histone chaperone complex, that inhibits cytoplasmic virus replication in insect and human cells. Using A51R proteins to probe FACT functions, we discovered that a post-translationally modified form of a FACT complex subunit is bound by A51R proteins and mislocalized to the cytoplasm during poxvirus infection. We also found several, unrelated RNA viruses to encode IEPs that prevent and/or reverse FACT subunit modification. Using virological, genetic, and biochemical approaches, we aim to reveal how viral IEPs impede FACT complex activity, the role of FACT in determining host susceptibility to infection, and the function(s) of FACT subunit post-translational modification in both antiviral and normal cellular processes. Finally, we are developing a pipeline to exploit our arbovirus-lepidopteran host system to identify additional IEPs targeting conserved host factors that act as key determinants of intracellular pathogen restriction. Using this pipeline, we identified ~10 IEPs encoded by bacterial pathogens infecting mammals that relieve arbovirus restriction in insect cells, suggesting these IEPs target host defenses that broadly restrict viruses and bacteria. Our overall mission is to identify novel IEPs encoded by mammalian pathogens in our unique system and use them to provide mechanistic insights into the antiviral (and normal) functions of the conserved host machinery they manipulate.